Abstract This PPG competing renewal application is driven by the fundamental and therapeutic importance of costimulatory pathways in regulating T cell activation, tolerance, and exhaustion and builds upon our significant progress since initial award of this PPG in 2003. Our productivity is highlighted by 111 primary publications and 22 reviews. Our PPG also has had a significant role in fostering development of junior faculty, and sharing novel mAbs and mouse strains with the broader scientific community, resulting in better understanding of costimulation above and beyond our PPG aims. Our overarching goal is to develop a comprehensive understanding of how positive and negative second signals regulate T cell activation, tolerance and exhaustion. Working together, we have discovered that the PD-1 pathway has multifaceted immunoregulatory functions. To better understand mechanisms of PD-1 signaling in vivo, we generated novel PD-1 signaling domain mutant mice and determined that both the PD-1 ITIM and ITSM motifs mediate PD-1 signaling in vivo. Notably, our data suggest the possibility to dissociate beneficial effects of PD-1 pathway blockade on pathogen/tumor immunity from autoimmunity and immunopathology. We also found that the inhibitory receptors CD101 and CD112R are highly expressed on more terminally exhausted T cells with distinctive functional properties during chronic infection, and on highly suppressive regulatory T cells. These discoveries demonstrate ongoing synergy within our PPG. The sharing of unpublished results and discussion of data have inspired hypotheses and experiments bidirectionally in all projects, which drive the focus of this application. Our major goals are to investigate: 1) Roles of the PD-1 ITSM and ITIM motifs in regulating different T cell subsets in different disease states, 2) How the PD-1 pathway controls pathogenic and protective T cells in tissue-dependent contexts, 3) Roles of CD101 and CD112R in controlling T cell activation, tolerance and exhaustion, and 4) Interactions of CD101 and CD112R with PD-1 in regulating T cell responses, using models of infection, chronic graft versus host disease (cGVHD), cancer and autoimmunity. Our proposed Program will consist of 3 highly integrated and interactive Projects, supported by 3 Cores. Project 1 (Sharpe/Ahmed) will focus on the roles of PD-1, CD101 and CD112R in controlling protective immunity during acute and chronic viral infections. Project 2 (Kuchroo/ Sharpe) will study how PD-1 and CD112R regulate autoimmunity and anti- tumor immunity. Project 3 (Blazar/Sage) will investigate how PD-1, CD101 and CD112R regulate cGVHD. Core A (Sharpe) will provide administrative and scientific coordination. Core B (Freeman) will provide novel mAbs and Ig fusion proteins. Core C (Sharpe) will provide novel mouse strains. The use of the same standardized tools makes it possible to compare and contrast results in different settings and disease models. Mechanistic insights from our proposed studies should guide translation into therapies targeting coinhibitory pathways for human chronic viral infections, cancer, autoimmune diseases and transplantation.